A genetic and biochemical analysis of cooperative protein-DNA interactions in bacteriophage HK022

Abstract

Cooperative binding of specific DNA-binding proteins to DNA plays a crucial role in gene regulation. To understand the molecular basis of cooperative protein-DNA interactions and their role in gene regulation, the lambdoid phage HK022 was used as a model system to study cooperative binding of HK022 CI repressor to DNA. HK022 CI repressor binds to two adjacent operators with a high degree of cooperativity. In this work, a combination of genetics and biochemistry was used to study cooperative binding of HK022 CI regressor by analyzing the effects of changing either the DNA binding sites or the protein on cooperativity. In the first part of this dissertation, the effect of changing the spacing between the two adjacent operators (O(R)1 and O(R)2) on cooperativity and on the conformation of the complex was examined. The highest cooperativity was observed with wild type spacing of 9 bp, implying that the wild type spacing confers the most favorable cooperative interaction. Considerable cooperativity was retained for most spacing variants, but was abolished when the operators lay on opposite faces of the DNA helix. Almost all spacing variants conferred changes in the conformation of the DNA-protein complex. The major conclusions from this study are that: (1) the protein-DNA complex is flexible enough to allow some cooperativity in most spacing variants; (2) a protein-DNA complex involving the same specific binding sites and the same protein molecules can adopt many different conformations, depending on the spacing between the binding sites. In the second part of this investigation, a genetic screen was developed to isolate HK022 repressor mutants which are defective in cooperative binding to adjacent operators, but are normal in binding to a single operator, so as to identify the amino acids in CI responsible for the cooperative interactions. Five mutants (IL153, RS225, RG225, FS233, and +QK) were isolated from this screen. The cooperativity parameter o for these mutant proteins (except IL153) was determined by in vitro DNase I footprinting assay. The results indicated that +QK was a relatively strong mutant, with a reduction of about 20 fold in o; the others were weaker, reducing o about 4 to 5 fold. Two double mutant combinations (+QK-RS225 and +QK-RG225) conferred greater cooperativity defects than the single mutants. One double mutant (RS225-IL153) restored nearly wild type cooperativity indicating that those two single mutations suppress each other perhaps by a lock-and-key mechanism. The cooperativity mutants were used to demonstrate the importance of cooperativity for HK022 phage immunity and the lysis-lysogeny decision.